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. 2026 Jun 4;46:102624. doi: 10.1016/j.bbrep.2026.102624

Salvianolic acid B mitigates senescence and promotes osteogenesis of senescent bone marrow mesenchymal stem cells via the PI3K/AKT pathway

Shaozi Zhong a,b, Dianxuan Wu a,b, Qize Chen a,b, Chun Zeng a,b,⁎⁎⁎, Huabin Chen a,b,⁎⁎, Jie Huang c,⁎
PMCID: PMC13254721  PMID: 42291502

Abstract

Senescence is a major contributor to osteoporosis, with progressive bone loss in later life characterizing age-related osteoporosis. The decline in bone marrow mesenchymal stem cells (BMSCs) function, particularly the reduction in osteogenic capacity, plays a central role in this process. Therefore, improving the function of senescent BMSCs could potentially slow the age-related osteoporosis progression. Salvianolic acid B (Sal-B), the most abundant and bioactive water-soluble compound derived from the traditional herb Salvia miltiorrhiza and growing evidence supports its efficacy against osteoporosis. However, the underlying cellular and molecular mechanisms remain unclear. This study aimed to investigate the effects and mechanisms of Sal-B on senescent BMSCs in vitro. Our study show that Sal-B not only promoted osteogenic differentiation but also attenuated cellular senescence in senescent BMSCs. Mechanistically, the PI3K/AKT pathway played a crucial role in mediating these beneficial effects. These findings clarify the biological action and mechanism of Sal-B at the cellular level, providing a theoretical foundation for subsequent in vivo studies.

Keywords: Salvianolic acid B, Bone marrow mesenchymal stem cell, Senescence, Osteogenic differentiation, PI3K/AKT pathway

Highlights

  • •

    Sal-B enhances osteogenic activity and attenuates cellular senescence in BMSCs.

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    Transcriptomics: PI3K-AKT drives senescent BMSC fate.

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    Sal-B partially restored the suppressed PI3K-AKT pathway in senescent BMSCs.

1. Introduction

Senescence is a multifactorial natural phenomenon characterized by declining organ function and homeostatic imbalance. These degenerative processes contribute to the development of age-related diseases, such as osteoporosis [1]. Osteoporosis is a systemic condition characterized by reduced bone mass and deteriorated mechanical strength, thereby elevating fracture risk [2]. With the global senescence population growing rapidly, the incidence of osteoporosis is also rising substantially, which creates a huge burden on public health [3].

Alterations in the quantity and functionality of BMSCs, particularly the decline in their osteogenic differentiation capacity, represent a key contributing factor to age-related osteoporosis [4,5]. Mounting evidence indicates that senescence impairs essential stem cell functions, including self-renewal, activation and differentiation potential [6]. Therefore, modulating the activity and osteogenic function of senescent BMSCs constitutes a promising therapeutic strategy for age-related osteoporosis [7].

Salvianolic acid B (Sal-B), one of the primary bioactive compounds derived from Salvia miltiorrhiza, exhibits potent antioxidant activity. It has been demonstrated to exert anti-apoptotic, anti-inflammatory, and anti-fibrotic effects, enhance stem cell proliferation and differentiation, and show therapeutic potential in cardiovascular diseases and liver fibrosis [8]. Nevertheless, growing evidence indicates that Sal-B also exerts significant beneficial effects against osteoporosis, revealing a newly recognized therapeutic potential[[9], [10], [11], [12]]. However, its precise efficacy and underlying mechanisms in age-related osteoporosis still unclear.

In this study, we established an in vitro senescence model of BMSCs to investigate the effects of Sal-B on cellular senescence and osteogenic capacity, and to elucidate the essential role of the PI3K/AKT pathway in Sal-B-mediated therapy. The findings provide a scientific foundation for the potential clinical application of Salvia miltiorrhiza in the prevention and treatment of age-related osteoporosis.

2. Materials and methods

2.1. Isolation and culture of BMSCs

In our previous study [13], the isolation procedure for bone marrow-derived mesenchymal stem cells (BMSCs) has been described in detail. And, the stem cell characteristics of rat BMSCs have been verified through trilineage differentiation assays and flow cytometric analysis in our previous study. In brief, male Sprague-Dawley rats (4 weeks old) were sacrificed by cervical dislocation. BMSCs were isolated from the bilateral femurs and tibias of the rats. Under sterile conditions, the ends of the bones were cut off, and the bone marrow was flushed out using a 1-mL syringe filled with Alpha-modified Eagle's medium (αMEM). The bone marrow was then centrifuged to remove the supernatant, and the resulting cell pellet was re-suspended in Alpha-modified Eagle's medium (αMEM) supplemented with 10% FBS and 1 × penicillin-streptomycin. After 48 h, non-adherent cells were removed. When the cells reached 100% confluence, they were sub-cultured, and the culture passage was increased to Passage 1 (P1).

To establish a replicative senescence model, BMSCs were continuously passaged in vitro. Cells at passage 3 were used as the young control group, while cells derived from the same source were passaged at 1:3 ratio to passage 6 to serve as the replicative senescence group.

BMSCs were cultured in stem cell complete culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37 °C with 5% CO2. The medium was changed every 2–3 days. For subculture, BMSCs were seeded into T25 flasks at a density of 4 × 105 cells/cm2. When the cell density reached approximately 85%, cellular senescence was induced by exposing the cells to 1.4 mM hydrogen peroxide (H2O2) for 2 h. Following treatment, the cells were washed three times with phosphate-buffered saline (PBS) and incubated overnight in fresh complete medium. To inhibit PI3K or AKT, 10 μM LY294002 (HY-10108; MCE, USA) was used. In the Sal-B treatment group, cells were treated with 100 μmol/L Sal-B for 7 days before protein or mRNA extraction, followed by Western blot or qPCR analysis.

2.2. CCK-8 assays

For the cell proliferation assay, BMSCs were randomly divided into control, senescence and senescence with Sal-B treatment group. The senescence with Sal-B treatment was further subdivided into four subgroups receiving 10, 50, 100 or 150 μmol/L Sal-B, respectively. Each group of BMSCs was seeded into 96-well plates at a density of 4 × 103 cells/well in 100 μL of cell suspension. To minimize edge effects, the peripheral wells were filled with an equal volume of sterile phosphate-buffered saline (PBS). After adding 10 μL of CCK-8 solution to each well, the plates were incubated in an incubator for 2 h. The optical density values of each well were measured at a wavelength of 450 nm using an enzyme-labeled instrument at 0, 1, 3, 5, and 7 days.

For the cytotoxicity assay, BMSCs were randomly divided into control and Sal-B treatment. The Sal-B treatment was further subdivided into four subgroups receiving 10, 50, 100 or 150 μmol/L Sal-B and treated for 3days. And all other experimental conditions and processes were kept consistent with the CCK-8 proliferation assay.

2.3. Alizarin Red staining

Cells from each group were seeded in 12-well plates, and osteogenic differentiation was induced using a commercially available human mesenchymal stem cell osteogenic induction kit. After 21 days of induction, the cells were fixed and stained with Alizarin Red. The calcium nodule formation was visualized and quantified using an inverted microscope.

2.4. SA-β-Gal staining

SA-β-galactosidase (SA-β-Gal) staining was performed to assess cellular senescence using a SA-β-Gal staining kit (C0602; Beyotime Biotechnology, China) as previously described [6,7]. Briefly, cella were fixed with 4% paraformaldehyde for 10 min and then washed three times with PBS, followed by incubation with SA-β-Gal solution at 37 °C overnight. Senescent BMSCs were identified by the presence of blue staining and visualized under a light microscope.

2.5. Immunofluorescence

BMSCs mounted on glass slides were washed three times with PBS and fixed with ice-cold 4% paraformaldehyde for 15 min, followed by another PBS wash. Thereafter, the cells were permeabilized with 0.25% Triton X-100 for 20 min and then blocked with goat serum for 1 h at room temperature, prior to incubating with primary antibodies (1:100: 28248-1-AP; Proteintech, U.S.) overnight at 4 °C. The following day, after washing three times with PBS, the cells were incubated with the corresponding fluorescent secondary antibodies (1:300: AS039; ABclonal, China) for 1 h at room temperature in the dark. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min at room temperature. Fluorescence images were captured using an Automated Inverted Microscope System (Olympus).

2.6. qRT-PCR

Total RNA was extracted from the cells in each group using an RNA extraction kit, with the concentration and purity of the extracted RNA being determined using a spectrophotometer. Genomic DNA contamination was removed by DNase I treatment, and cDNA was synthesized from 1 μg of total RNA using a reverse transcription kit. Quantitative real-time PCR (qRT-PCR) was performed using the synthesized cDNA as a template and gene-specific primers (listed in Supplementary Table S1). The relative expression levels of target genes were calculated using the 2−ΔΔCT method, with GAPDH serving as the internal reference gene.

2.7. Western blotting

Proteins were separated using 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% skimmed milk in TBST for 1 h at room temperature. After blocking, the membranes were incubated overnight with primary antibodies at 4 °C. The following day, the membranes were incubated with the corresponding secondary antibodies at room temperature for 1 h. Protein bands were visualized using ECL developer, and the relative grayscale values of the protein bands were quantified using ImageJ software.

The following primary antibodies were used: anti-P21 (1:2000: 28248-1-AP; Proteintech, U.S.), anti-P16 (1:2000: 10883-1-AP; Proteintech, U.S.), anti-ALP (1:2000: YM8317; Immunoway, U.S.), anti-Runx2 (1:2000: YM8347; Immunoway, U.S.), anti-PI3K (1:1000: YM3503; Immunoway, U.S.), anti-Phospho-PI3K (1:1000: YP0224; Immunoway, U.S.), anti-AKT (1:1000: YT0185; Immunoway, U.S.), anti-Phospho-AKT (1:1000: YP0006; Immunoway, U.S.), and anti-β-Actin (1:5000: 66009-1-Ig; Proteintech, U.S.).

2.8. Statistical analysis

All statistical analyses were performed using SPSS 19.0 software (SPSS Inc., Chicago, USA). Data are expressed as the means ± standard deviation. One-way analysis of variance was used for comparisons among multiple groups, and the LSD t-test was used for comparison between two groups. Statistical significance was set at P < 0.05.

2.9. Bioinformatics analysis

Transcriptomic profiling data were processed and analyzed using R (version 4.5) with key Bioconductor packages. Differential expression analysis was subsequently conducted using DESeq2. Visualization of differentially expressed genes (DEGs) included volcano plots (ggplot2) and hierarchical clustering heatmaps (pheatmap) of Z-score normalized expression values. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed based on DEGs. All statistical analyses and visualizations were implemented in RStudio.

3. Results

3.1. The dose-dependent effect of Sal-B on the proliferation of senescent BMSCs

The results of the CCK-8 assay demonstrated that the viability of BMSCs was significantly decreased after senescence induce (P < 0.05; Fig. 1A). Treatment with Sal-B markedly enhanced the proliferation capacity of senescent BMSCs in a dose-dependent manner within the concentration range of 0-100 μmol/L (P < 0.05). Fig. 1B illustrates the cytotoxic effects of Sal-B on BMSCs across different concentrations. Within the range of 10–150 μmol/L, no toxic effect was observed across the concentration.

Fig. 1.

Fig. 1

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The effect of salvianolic acid B on the proliferative capacity and cytotoxicity of BMSCs by CCK-8 (n = 3).

Cellular senescence is a major factor leading to impaired proliferative activity. And the mechanisms of Sal-B against cellular senescence have been well established that related to the inhibition of MAPK and NF-κB signaling pathways [14,15]. Here, we further demonstrate that Sal-B restores the proliferative capacity of senescent BMSCs.

3.2. The effects of Sal-B on the osteogenic expression and differentiation of senescent BMSCs

Alizarin Red staining revealed a significant reduction in calcium nodule formation in senescent BMSCs compared to the control group (Fig. 2A). In contrast, treatment with Sal-B increased the intensity of Alizarin Red staining, indicating enhanced calcium nodule formation in senescent BMSCs. Western blot further confirmed that Sal-B treatment enhanced the osteogenic capacity of senescent BMSCs, as demonstrated by increased protein expression of the osteogenic markers Runx2 and ALP (Fig. 2B–D).

Fig. 2.

Fig. 2

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The effects of Sal-B on the osteogenic expression and differentiation of senescent BMSCs. (A) Osteogenic differentiation of BMSCs detected using Alizarin Red staining (Sal-B concentration: 100 μmol/L; n = 3; the scale bar represents 100 μm). (B) Western blot of Runx2 and ALP proteins in BMSCs of the different treatment groups. (C-D) Quantitative data for relevant protein expression normalized to β-Actin. (E-H) The mRNA expression of osteogenic differentiation-related factors in senescent BMSCs by qRT-PCR (Sal-B concentration: 100 μmol/L; n = 3). ∗: P < 0.05; #: P < 0.01.

Next, we analyzed the mRNA expression of osteogenic markers, including ALP, Runx2, OCN, and BMP4. Compared to the control group, the expression levels of these osteogenic genes were significantly downregulated in the senescence group (P < 0.05; Fig. 2E–H). However, Sal-B treatment significantly upregulated the expression of these genes compared to the untreated senescence group (P < 0.05), although the expression levels remained lower than those in the control group (P < 0.05).

Runx2 is a key transcription factor that induced the osteogenic differentiation of BMSCs [16]. It drives the expression of genes involved in bone matrix formation, such as BMP4, thereby promoting bone generation [17]. BMP4 enabled increase the expression of Runx2, thereby further stimulating bone formation. ALP and OCN are key marker genes of osteoblasts, promoting cellular maturation and calcification by hydrolyzing various phosphates [18]. Our findings indicate that Sal-B significantly improves the osteogenic capacity of senescent BMSCs. And suggest that Sal-B effectively improves the osteogenic function of senescent BMSCs, demonstrating the potential therapeutic agent for age-related osteoporosis.

3.3. The effects of Sal-B on senescence, proliferation, and senescence-related secretory factor expression of BMSCs

We next investigate the effect of Sal-B on the senescence process of BMSCs. Results show that compared to control group, the proportion of SA-β-Gal-positive cells was significantly increased in senescence group. In contrast, Sal-B treatment significantly reduced the proportion of SA-β-Gal-positive cells compared to the untreated senescence group (P < 0.01; Fig. 3A and B).

Fig. 3.

Fig. 3

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The effect of Sal-B on senescence, proliferation, and senescence-related secretory factor expression of senescent BMSCs. (A) SA-β-Gal staining revealing the presence of blue-stained senescent cells (the scale bar represents 20 μm). (B) The ratio of SA-β-Gal-positive cells to the total number of cells. (C) Immunofluorescence staining for P21 (the scale bar represents 20 μm). (D) The ratio of P21-positive cells to the total number of cells. (E) Western blot of P21 and P16 proteins in BMSCs of the different treatment groups. (F) Quantitative data for relevant protein expression normalized to β-Actin. (H-J) The effects of Sal-B on the expression of senescence-related secretory factors in senescent BMSCs determined using qRT-PCR (Sal-B concentration: 100 μmol/L; n = 3). ∗: P < 0.05, #: P < 0.01.

Additionally, Sal-B treatment led to a significant decrease in the proportion of P21-positive cells (P < 0.01; Fig. 3C and D). Western blots show that Sal-B treatment significantly reduced the expression of the senescence-associated proteins P21 and P16 (P < 0.01; Fig. 3E–G). Collectively, these findings indicate that Sal-B mitigates BMSC senescence and contributes to maintenance of the proliferative capacity of these cells.

Furthermore, we analyzed the expression of the senescence-related secretory factors (SASPs) IL-6, TNF-α, and MMP1. Accordingly found that, compared to the control group, these genes were significantly increased in senescent BMSCs. However, Sal-B treatment downregulated their expression compared to the untreated senescence group (P < 0.05; Fig. 3H–J), although the levels remained higher than those in the control group.

It is well established that cellular senescence is a key factor leading to the decline in both stemness and osteogenic function of BMSCs, and impairs the osteogenic differentiation. [19,20]. In addition, senescent cells secrete inflammatory cytokines, chemokines, and proteases, collectively termed the senescence-associated secretory phenotypes (SASPs) [21], which drives senescence signals to neighboring normal cells in a paracrine manner [22,23]. Slowing or reversing the senescence process in BMSCs represents a viable therapeutic strategy for age-related osteoporosis [24]. Our results indicate that Sal-B effectively alleviated cellular senescence in BMSCs and suppressed the expression of SASP-related genes.

3.4. Sal-B Exerts the Therapeutic Effects by modulating the PI3K/AKT pathway

To further investigate the key alterations occurring in senescent BMSCs, we analyzed the data for human BMSCs from the GEO database [25]. Cells cultured in vitro at passage 3 (P3) served as the control group, while cells at passage 6 (P6) constituted the senescence group. Differential expression analysis identified 852 downregulated genes and 474 upregulated genes (p < 0.05, |logFC| > 0.585) (Fig. 4A and B).

Fig. 4.

Fig. 4

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Bioinformatic Analysis Reveals the Transcriptomic Alteration in Senescent BMSCs. (A) Volcano plot of DEGs. (B) Hierarchical clustering of DEGs. (C) GO enrichment of DEGs. (D) KEGG enrichment of DEGs.

To further confirm that P6 BMSCs, in the transcriptomic data, had entered a senescent state, we performed GSEA on the transcriptomic dataset, along with statistical analysis of senescence-associated gene expression (Fig. S2). GSEA analysis revealed that senescence-associated gene sets (e.g., P53 Pathway, Replicative Senescence, Apoptosis) were activated in P6 BMSCs, whereas proliferation-associated gene sets (e.g., E2F Target Genes, G2M Checkpoint, and Cell Cycle) were markedly suppressed (Fig. S2A). To directly verify the senescent state of P6 BMSCs in our used dataset, we performed a statistical analysis of the expression of senescence-associated genes (CDKN1A, CDKN2A, and TP53) in P3 versus P6 BMSCs (Fig. S2B). The results showed that expression of these genes was significantly higher in P6 BMSCs compared with P3 (p < 0.05). Based on the above studies and results, we are confident that P6 cells have indeed entered a senescent state.

GO enrichment analysis revealed the differentially expressed genes (DEGs) were significantly enriched in biological processes and cellular components related to mitotic chromosome segregation and regulation. These included processes such as condensed chromosome, mitotic and chromatic segregation and involved key molecular functions like microtubule binding and motor activity. Additionally, a subset of genes also showed enrichment in extracellular matrix regulation (Fig. 4C). To further explore the underlying molecular mechanisms, we performed KEGG pathway enrichment analysis. The results demonstrated significant enrichment in the PI3K-AKT signaling pathway (Fig. 4D). And the GSEA analysis indicated that the PI3K/AKT-related signaling pathways (e.g., PI3K-AKT-mTOR pathway, PI3K pathway) was significantly suppressed in P6 BMSCs (Fig. S2A). This finding is consistent with previous reports showing that PI3K/AKT pathways were significantly inhibited in senescent BMSCs [26,27], and given its established role in regulating osteogenic differentiation [8,9], such inhibition may critically contribute to the pathogenesis and progression of osteoporosis.

To further examine PI3K-AKT pathway alterations in senescent BMSCs and assess whether Sal-B reverses these changes, we performed Western blot analysis, which confirmed that the expression levels of PI3K, AKT, and their phosphorylated forms were significantly reduced in senescent BMSCs compared to the control group. In contrast, Sal-B treatment significantly upregulated the expression of these pathway proteins compared to the untreated senescence group (P < 0.01; Fig. 5A–E). And we further performed qPCR analyses to measure the mRNA expression levels of PIK3CA and AKT1, which encode key positive regulatory components of PI3K/AKT pathway. And we found that Sal-B significantly upregulated the expression of both genes at the transcriptional level (P < 0.01; Fig. 5F and G). These results suggest that the osteogenic effects of Sal-B on senescent BMSCs may be mediated through the stabilization of the PI3K/AKT signaling pathway.

Fig. 5.

Fig. 5

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Sal-B Exerts the Therapeutic Effects by Modulating the PI3K/AKT Pathway. (A) Western blot of PI3K, p-PI3K, AKT, and p-AKT proteins in the BMSCs of the different treatment groups. (B-E) Quantitative data for relevant protein expression normalized to β-Actin. (F and G) The mRNA expression of PIK3CA and AKT1 in BMSCs by qRT-PCR. (I) Western blot of ALP, Runx2, P21 and P16 proteins in BMSCs of the different treatment groups. (Sal-B concentration: 100 μmol; LY294002 concentration: 10 μM; n = 3). (J-M) Quantitative data for relevant protein expression normalized to β-Actin. ∗: P < 0.05; #: P < 0.01.

To further investigate the necessity of the PI3K/AKT pathway in the therapeutic effect of Sal-B, we conducted a rescue experiment using the PI3K inhibitor LY294002. The results showed that, consistent with our previous findings, Sal-B treatment significantly ameliorated BMSCs senescence and promoted the osteogenic phenotype. However, the treatment of the PI3K inhibitor LY294002 markedly abolished these therapeutic effects of Sal-B. These findings further confirm that Sal-B improves BMSCs senescence and osteogenic differentiation by activating the PI3K/AKT pathway (P < 0.05; Fig. 5I–M).

We further performed rescue experiments with Alizarin Red staining to further validate the therapeutic mechanism of Sal-B. The Alizarin Red staining results indicated that LY294002 treatment significantly inhibited the therapeutic effect of Sal-B on the osteogenic function of senescent BMSCs. Meanwhile, LY294002 treatment also reversed the alleviation of the senescence process in BMSCs by Sal-B (Fig. 5H).

The PI3K/AKT pathway has been demonstrated to play a critical role in both osteogenic regulation and the senescence process of MSCs[[28], [29], [30], [31]]. It modulates the balance between osteogenesis and adipogenesis in BMSCs, considered a critical therapeutic target for osteoporosis [32]. In the processes of senescence, PI3K/AKT pathway regulates senescence by various downstream factors, such as mTOR signaling and the P53/P21 axis [30,33].

In this study, we demonstrate that the PI3K/AKT pathway serves as a critical role in Sal-B therapeutic effects in senescent BMSCs. This elucidates, at the molecular mechanistic level, how Sal-B alleviates BMSC senescence and enhances osteogenic differentiation and providing a novel theoretical foundation for age-related osteoporosis.

4. Discussion

Senescence and age-related diseases are pernicious worldwide problems, yet effective solutions remain to be elucidated. As a result, research on senescence and anti-senescence strategies has garnered considerable attention. Bone marrow mesenchymal stem cells play key roles in maintaining the balance between bone formation and resorption, in which the differentiation of BMSCs to osteoblasts serves as the primary source of new bone regeneration. However, during senescence, the osteogenic differentiation capacity of BMSCs progressively declines, leading to an imbalance between bone formation and resorption and increasing the risk of osteoporosis and other age-related bone disorders. Accordingly, efficient regulation of the senescence of BMSCs is one of the particular importance for maintaining bone homeostasis. In this study, in which we used an in vitro model of BMSCs senescence, we investigated the effects of plant constituent Sal-B on senescent BMSCs and confirmed that this compound can inhibit senescence and the expression of cell senescence-related secretory factors in senescent BMSCs, thereby promote the proliferation and osteogenic differentiation of these senescing BMSCs.

Senescence is a progressive pathophysiological process that characterized by gradual declines in physiological function and proliferative, which collectively play important roles in maintaining normal tissue homeostasis [34]. As a prominent risk factor, senescence contributes significantly to osteoporosis that characterized by reduced bone mass, deterioration of bone microarchitecture, and consequent fragility fractures. During the senescence of BMSCs, cells exhibit reduced activity and aberrant differentiation. Notably, the imbalance between osteogenic and adipogenic differentiation is considered a primary contributing factor to osteoporosis [35]. In this context, Qiao et al. [36] have confirmed in vitro that by stimulating the activity of osteoblasts and the expression of bone formation-related genes, Sal-B can protect osteoblasts treated with prednisolone acetate, suggesting that Sal-B can combat the occurrence of plasmic osteoporosis. In further studies, Wang et al. [37] demonstrated that by loading large doses of Sal-B, silk fibroin/graphene oxide scaffolds can promote a significant enhancement of the osteogenic differentiation of rat BMSCs, enhance the migration of endothelial cells and the formation of tubules in vitro, and promote bone regeneration in rat skull defect models. These finding indicate that graphene oxide can serve as an effective slow-release carrier for therapeutic plant molecules such as Sal-B that can contribute to the repair bone defects by promoting osteogenic differentiation and angiogenesis. Our study further demonstrated that Sal-B effectively promotes the osteogenic differentiation of senescent BMSCs, indicating that it not only activates osteogenesis in healthy BMSCs but can also restore the impaired osteogenic function in senescent BMSCs. These findings highlight the considerable potential of Sal-B as an anti-osteoporotic agent.

Recent studies on the mechanisms of cellular osteogenic differentiation have revealed that the PI3K/AKT pathway plays a central role in the regulation of this process [[38], [39], [40], [41]]. For instance, Chen et al. [42] demonstrated that graphene oxide/black phosphorus-functionalized collagen scaffolds enhance the osteogenic differentiation capacity of BMSCs, an effect that was reversed by PI3K/AKT pathway inhibition, underscoring the critical role of this pathway in BMSC osteogenesis. Consistent with these findings, our results revealed that Sal-B upregulates the expression of PI3K/AKT pathway proteins in senescent BMSCs, suggesting that the osteogenic effects of Sal-B likely involved in the stabilization of PI3K/AKT signaling.

Elucidating the mechanisms associated with senescence is a particularly active area of research in academic and biomedical fields. In 1956, Denham Harman proposed the free radical theory of senescence, suggesting that the degenerative changes in senescence are mediated by the harmful effects of free radicals produced during normal cellular metabolism [43]. Subsequent studies have confirmed that chronic oxidative stress, driven by elevated levels of reactive oxygen species (ROS), contributes to cellular senescence [44]. For example, Fei et al. [45] demonstrated that downregulation of the histone acetyltransferase KAT6A in senescent BMSCs leads to intracellular ROS accumulation, resulting in reduced stemness and impaired osteogenic differentiation. Sal-B, with its molecular structure containing nine phenolic hydroxyl groups, exhibits potent antioxidant properties through the donation of multiple hydrogen atoms. Previous studies have indeed confirmed that Sal-B has strong free radical scavenging and antioxidant capacities [17,18], and regulates antioxidant enzyme expression by upregulating SOD, GSH-Px, and HO-1 while inhibiting NOX-2 and NOX-4 [46]. Moreover, Sal-B inhibits poly (ADP-ribose) polymerase 1 (PARP-1) activity, preventing NAD + depletion and upregulating Grx1 expression, thereby reducing ROS production and lipid peroxidation products such as malondialdehyde [47]. In addition, Sal-B has been shown to induce the down-regulated expression of Keap3 and the up-regulated expression of Nrf3 via the NAD-dependent deacetylase Sirtuin 1 (SIRT2) and the phosphatidylinositol 1-kinase (PI1K)/Akt signaling pathway, thereby promoting the production of antioxidant enzymes [48]. Yang et al. [49] also reported that Sal-B protects endothelial cells from oxidative stress-induced damage by downregulating NADPH oxidase expression, thereby inhibiting apoptosis. The H2O2-induced senescence model is largely based on oxidative stress and DNA damage mechanisms. To distinguish between acute oxidative toxicity and cellular senescence, we performed qPCR cross-validation using a replicative senescence model (Fig. S1). The results demonstrated that Sal-B also exhibited marked therapeutic efficacy in the replicative senescence model, suggesting that its therapeutic mechanism may extend beyond mere peroxide scavenging. Mechanistically, the PI3K/AKT pathway plays a critical role in Sal-B treatment, mediating not only the improvement of osteogenic function but also the alleviation the processes of senescence in BMSCs.

However, it is important to note that translating these findings into animal models or clinical applications will require tailored formulation optimization and pharmacokinetic studies to achieve an effective and safe concentration range. This necessary step, along with further in vivo validation, represents a current limitation of our study.

In summary, this study clearly establishes the biological effects and mechanisms of Sal-B at the cellular level. These findings position Sal-B as a promising candidate for alleviating age-related osteoporosis, while also providing a solid theoretical foundation for subsequent in vivo research.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Funding

This work was supported by the following funding resources: the GuangDong Basic and Applied Basic Research Foundation (2022A1515110910), the Medical Scientific Research Foundation of Guangdong Province (A2023105) and the President Foundation of The Third Affiliated Hospital of Southern Medical University (YQ2021009), the Research Project of Guangdong Provincial Bureau of Traditional Chinese Medicine (NO.20222083).

CRediT authorship contribution statement

Shaozi Zhong: Formal analysis, Software, Visualization, Writing – original draft. Dianxuan Wu: Data curation, Formal analysis, Methodology, Visualization. Qize Chen: Data curation, Methodology. Chun Zeng: Project administration, Resources, Supervision, Writing – review & editing. Huabin Chen: Funding acquisition, Project administration, Resources, Writing – review & editing. Jie Huang: Conceptualization, Data curation, Funding acquisition, Project administration, Resources, Visualization, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

Not applicable.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrep.2026.102624.

Contributor Information

Chun Zeng, Email: zengdavid@126.com.

Huabin Chen, Email: sportsmed-chen@foxmail.com.

Jie Huang, Email: huangjie-dentist@gzucm.edu.cn.

Appendix A. Supplementary data

Figure S1. The mRNA expression of CDKN1A, CDKN2A, ALP, RUNX2, PIK3CA and AKT1 in replicative senescence BMSCs by qRT-PCR (n = 3).

Figure S2. The GSEA analysis and statistical analysis of senescence-associated gene expression in the transcriptomic of human BMSCs (P3 vs P6). (A) GSEA analysis of transcriptomic in human BMSCs. A positive NES indicates that the corresponding function or pathway is activated in P6 BMSCs, whereas a negative NES indicates inhibition. (B) Statistical analysis of senescence-associated gene expression (CDKN1A, CDKN2A and TP53) from transcriptomic in human BMSCs. ∗: P < 0.05.

The following are the Supplementary data to this article.

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Data availability

Data will be made available on request.

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